Hello all,
I am trying to induce NET induction by IL-8 (100 ng/ml) using healthy neutrophils and patients' neutrophils. I quantify the NET induction by Picogreen assay.
In case of healthy neutrophils, i could not get an significant increase in NET induction by IL-8. However, several articles reported that they could induce NET formation by 50-100 ng/ml IL-8 using neutrophils from healthy donors.
Do you have any idea why we could not replicate their experiments using healthy neutrophils?
Thank you very much.
Hello Le-Duy Pham:
PMA can induce NET formation in the nano-micro molar range. But maybe the method you are using is not the best. To my knowledge immunofluorescence is better, given that not all DNA is liberated and becomes soluble to be detected by the picogreen assay. Also changes in morphology are indicative of the netosis induction process and with different inductors the time of netosis induction might be different. Regarding your question of replication, there are several factors that i would consider (origin of IL-8 such as brand, recombinant?, tested for activity and signaling?)
in RA it has been described that neutrophils from patients have increased susceptibility to dye by netosis than does healthy controls and it has been suggested that these differences might be related to PKC activity.
My recommendations:
1) Use PMA as a control, 2)Try immunofluorescence with DAPI and MPO or DAPI alone.
Best regards.
Thank you Julio Enrique,
I used PMA as positive control and it worked with picogreen assay...
Hello Le-Duy Pham:
Have the other groups used a priming step with healthy controls (usually a short stimulation, 10-15 minutes, with TNF before stimulation with LPS)? I have found that neutrophils from healthy controls do not undergo NETosis in response to IL-8 or LPS without a priming step before.
I would also recommend using some basic microscopy to evaluate the morphology of the neutrophils following stimulation with IL-8 to see if there is any difference.
Best regards,
Rob
Hello Robert,
After treating neutrophils with IL-8, I observed a significantly change in its morphology (from round shape to an star shape), could be due to an activation.
May I ask how you measured NET production? by picogreen assay or by confocal microscopy?
Thank you,
Duy
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